Tryptophan synthase and tryptophanase catalyze the condensation of indole and L-serine to form L-tryptophan; in addition, tryptophanase catalyzes the cleavage of L-tryptophan to indole, pyruvic acid, and ammonia. Oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan, which are analogs of the indolenine tautomer of L-tryptophan, a proposed reaction intermediate, are potent competitive inhibitors of these enzymes (Phillips, R.S., Miles, E.W., and Cohen, L.A. (1984) Biochemistry 23, 6228-6234). These inhibitors can exist in diastereoisomeric forms due to the asymmetric center at C-3 of the heterocyclic rings. We have separated the diastereoisomers of 2,3-dihydro-L-tryptophan by preparative reverse-phase HPLC: these diastereoisomers are designed "A" and "B" in their order of elution from the HPLC column. Diastereoisomer B is a potent competitive inhibitor of tryptophan synthase; addition of diastereoisomer B to solutions of tryptophan synthase results in a new visible absorption peak at 494 nm, which is attributed to the alpha-carbanion of the amino acid-pyridoxal phosphate Schiff's base complex. In contrast, diastereoisomer A is a weak inhibitor and ligand for tryptophan synthase. With tryptophanase, the specificity is reversed: diastereoisomer A is a potent slow-binding inhibitor, whereas diastereoisomer B is a very poor inhibitor. The strict diastereospecificity exhibited by these enzymes provides strong support for the proposal that the indolenine tautomer of L-tryptophan is an intermediate in the reactions of both enzymes. Furthermore, the specificity for different diastereoisomers suggests that these enzymes catalyze their reactions via enantiomeric indolenine intermediates.